Understanding Env Function in HIV-1 Infection: The Design, Synthesis and Validation of Small Molecule HIV-1 Env Inhibitors Project 3: Summary: The Central Goal of this Program Project (P0-1-GM-06550) comprises the identification and understanding of the functional states of the HIV-1 envelope (Env) trimer: (a) in the standalone virion; (b) during the HIV-1 cell entry process; and (c) during the process of antibody dependent cellular cytotoxicity (ADCC) of HIV-1 infected cells. Specifically we propose to design, synthesize and validate small molecules that bind to the HIV-1 Env glycoproteins (gp120 and gp41), and in turn modulate the HIV-1 Env functional states. Such an objective will require the accumulation of knowledge regarding the nature and function of the Env trimer at the highest possible level of atomic resolution. The derived understanding in turn will permit the design and synthesis of effective HIV-1 virion inactivators, cell entry inhibitors and ADCC sensitizers of HIV-1 infected cells, which hold the promise for both the prevention and eradication of HIV/AIDS. The specific targeted compounds, anticipated to bind the CD4 binding pocket, to other regions of the gp120 subunit and/or to the gp41 subunit, will arise through the close collaborative use of smFRET studies (Program 1), crosslinking-mass spectrometry, inhibitor binding bioassay data (Projects 2, 4 and Core B), high-resolution X-ray/cryoEM structures (Project 5 and collaborator Pamela Bjrkman) and computational modeling (Core A). Importantly, the recent technological advances in cryoEM and smFRET trimer visualization, in conjunction with biological measurements (Projects 1, 2, 4 and Core B), will permit elucidation of the specific effects that small molecule Env modulators have on the function of the HIV-1 Env. As such, the efforts of the P01 will clearly aid in the design and validation of new pharmacological tactics to address the AIDS pandemic. Project 3, The Synthetic Thrust of this Program Project, will focus specifically on the Program Project goals by developing and validating new classes of small molecules that interact uniquely with the HIV-1 Env trimer (gp120 and gp41), thereby providing insights into the landscape of Env conformations (i.e., Env states) and their biological implications. The close collaboration that has and will continue to occur between each of the five Program Projects and two Cores will clearly enhance the discovery, design, and validation of more highly functional, broad spectrum small molecule inhibitors/ADDC sensitizers of HIV-1 infected cells, as well as lead to labeled probes to identify, stabilize and/or modulate the important Env conformational changes leading to cell entry and/or infected cell eradication. Thus, through high throughput screens, computational modeling, design, and synthesis, along with structural analysis (smFRET/crystal/cryoEM) and bioassay/binding validation measurements, Project 3 will play a central, highly focused role in this Program Project.